Detection of viable but non-culturable staphylococci in biofilms from central venous catheters negative on standard microbiological assays

none7To our knowledge this is the first evidence that S. epidermidis and S. aureus can enter the VBNC state. If positively documented, their ability to persist in this state could help to explain the well-known prevalence of both species in recurrent CVC-related infections [4,8,17,18]. Detection of S. epidermidis in 62% of CVCs demonstrated to contain non-culturable bacteria (removed from febrile patients, 27% of whom also had a positive blood culture) supports this hypothesis. Further research into the relationship among VBNC forms found in CVC-associated biofilms, their actual metabolic activity and recurrent infection, as well as careful recording of concurrent clinical data, are required to gain insights into the possible role of VBNC bacterial cells in indwelling medical device-related persistent infections.G. Zandri;S. Pasquaroli;C. Vignaroli;S. Talevi;E. Manso;G. Donelli;F. BiavascoG., Zandri; Pasquaroli, Sonia; Vignaroli, Carla; S., Talevi; E., Manso; G., Donelli; Biavasco, Francesc

Erythromycin- and copper-resistant Enterococcus hirae from marine sediment and co-transfer of erm(B) and tcrB to human Enterococcus faecalis.

An erythromycin-, copper- and cadmium-resistant isolate of Enterococcus hirae from marine sediment was shown to harbor the plasmid pRE25 and to co-transfer erm(B) and tcrB to Enterococcus faecalis JH2-2. These data highlight the scope for antibiotic resistance selection by the marine environment through heavy metals and its possible involvement in antibiotic-resistant enterococcal infections

Antibiotic pressure can induce the viable but nonculturable state in Staphylococcus aureus growing in biofilms

Objectives: Staphylococcal biofilms are among the main causes of chronic implant-associated infections. We have recently suggested that their transformation into viable but non-culturable (VBNC) forms (i.e. forms capable of resuscitation) could be responsible for the recurrent symptoms. This work aims to establish whether Staphylococcus aureus biofilms can give rise to VBNC forms capable of being resuscitated in suitable environmental conditions, the role of different stressors in inducing the VBNC state and the conditions favouring resuscitation. Methods: S. aureus 10850 biofilms were exposed to different concentrations of antibiotic (vancomycin or quinupristin/ dalfopristin) and/or to nutrient depletion until loss of culturability. The presence of viable cells and their number were examined by epifluorescence microscopy and flow cytometry. Gene expression was measured by real-time PCR. Resuscitation ability was tested by growth in rich medium containing antioxidant factors. Results: Viable subpopulations were detected in all non-culturable biofilms. However, viable cell numbers and gene expression remained constant for 150 days from loss of culturability in cells from antibiotic-exposed biofilms, but not in those that had only been starved. Resuscitation was obtained in rich medium supplemented with 0.3% sodium pyruvate or with 50% filtrate of a late-log culture. Conclusions: Our findings demonstrate that S. aureus can enter the VBNC state in infectious biofilms. The presence of vancomycin or quinupristin/dalfopristin can inadvertently induce a true VBNC state or its persistence in S. aureus cells embedded in biofilms, supporting previous findings on the role of staphylococcal biofilms in recurrent infection

Aquaculture can promote the presence and spread of antibiotic-resistant Enterococci in marine sediments

Aquaculture is an expanding activity worldwide. However its rapid growth can affect the aquatic environment through release of large amounts of chemicals, including antibiotics. Moreover, the presence of organic matter and bacteria of different origin can favor gene transfer and recombination. Whereas the consequences of such activities on environmental microbiota are well explored, little is known of their effects on allochthonous and potentially pathogenic bacteria, such as enterococci. Sediments from three sampling stations (two inside and one outside) collected in a fish farm in the Adriatic Sea were examined for enterococcal abundance and antibiotic resistance traits using the membrane filter technique and an improved quantitative PCR. Strains were tested for susceptibility to tetracycline, erythromycin, ampicillin and gentamicin; samples were directly screened for selected tetracycline [tet(M), tet(L), tet(O)] and macrolide [erm(A), erm(B) and mef] resistance genes by newly-developed multiplex PCRs. The abundance of benthic enterococci was higher inside than outside the farm. All isolates were susceptible to the four antimicrobials tested, although direct PCR evidenced tet(M) and tet(L) in sediment samples from all stations. Direct multiplex PCR of sediment samples cultured in rich broth supplemented with antibiotic (tetracycline, erythromycin, ampicillin or gentamicin) highlighted changes in resistance gene profiles, with amplification of previously undetected tet(O), erm(B) and mef genes and an increase in benthic enterococcal abundance after incubation in the presence of ampicillin and gentamicin. Despite being limited to a single farm, these data indicate that aquaculture may influence the abundance and spread of benthic enterococci and that farm sediments can be reservoirs of dormant antibiotic-resistant bacteria, including enterococci, which can rapidly revive in presence of new inputs of organic matter. This reservoir may constitute an underestimated health risk and deserves further investigation